The D1 receptor (D1R) and D5R regulate ion transport in the renal proximal tubule, thick ascending limb, and other nephron segments. In the previous cycle, we showed that that SNX5 and SNX1 are important in the internalization of D1R and D5R, respectively. Moreover, selective renal deletion of SNX1 or SNX5 increases blood pressure in mice and rats. In human renal proximal tubule cells, SNX5 is expressed in non-lipid rafts while D1R, GRK4, and PP2A-catalytic subunit/ PPP2R2C2, and VPS 26 are in lipid and non-lipid rafts. D1-like receptor stimulation shifts D1R, GRK4, PP2A catalytic subunit/PPP2R2C2, and VPS 26 to non-lipid rafts where SNX5 is located. We hypothesize that the assembly of D1R with the signaling complex in membrane microdomains is governed by SNX5. SNX5 may act as a chaperone in the intracellular trafficking of D1R after agonist stimulation. This hypothesis will be tested in human renal proximal tubule cells; mouse proximal and distal convoluted tubule cells will be studied also because the role of these proteins on renal function and blood pressure will be tested in genetically manipulated mice. Specific aim 1 will test the hypothesis that SNX5 is important in the assembly of D1R, PP2A complex, and GRK4 in non-lipid raft membrane microdomains, as well as the internalization of this complex after agonist stimulation and subsequent recycling. Specific aim 2 will test the hypothesis that D1R is linked to adenylyl cyclase (AC) III and AC VI and D5R is linked to AC IV in specific membrane microdomains in renal proximal tubules. In non-renal proximal tubule cells, D1R is linked to AC V. However, AC V is not expressed in renal proximal tubules. Specific aim 3 will test the hypothesis that PPP2R2C, SNX1, and SNX5 are important in the regulation of blood pressure. These in vitro and in vivo studies may give important information on how specificity is conferred on G protein-coupled receptor action, related to second messengers, sodium transport, and subsequently hypertension. At least two hypotheses are novel: 1) that SNX5 is important in the assembly and signaling of D1R, PP2A, AC III and AC VI and GRK4 while D5R is linked to AC IV. At least two methods are novel: selective renal gene silencing in mice and inducible/reversible knockout.